Efficacy and sequence specific behavior of antisense compounds in biological systems depend upon a variety of factors, which include their resistance to enzymatic degradation, binding affinity for the target, susceptibility to RNase H cleavage when bound to a target mRNA and efficiency of cellular uptake. In order to achieve the proper balance of these features for efficient modulation of target gene expression, chemical modifications are made to the antisense compound. For example, unmodified phosphodiester antisense oligonucleotides are degraded rapidly in biological fluids containing hydrolytic enzymes (Shaw et al., Nucleic Acids Res. 1991, 19, 747-750; Woolf et al., Nucleic Acids Res. 1990, 18, 1763-1769) and first generation modified antisense compounds (i.e. 2′-deoxyphosphorothioate oligonucleotides) also are subject to activity-limiting degradation (Maier et al., Biomed. Pept., Proteins Nucleic Acids 1995, 1, 235-241; Agrawal et al., Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 7595-7599). Thus, modifications that render the oligonucleotide more resistant to nuclease activity are desirable in order to enhance antisense activity of the compound. Sugar moieties of antisense compounds also have been modified to increase such properties as lipophilicity, binding affinity for the target mRNA, chemical stability and nuclease resistance.
Distribution to peripheral tissues and ultimate uptake into the cells of target organs also is critical to the effectiveness of antisense compounds for treatment of a wide range of diseases. The highest concentrations of antisense compounds are typically found in the liver, kidney, spleen and lymph nodes, but can be detected in nearly all organs except for the brain (Geary et al., Curr. Opin. Investig. Drugs, 2001, 2, 562-573; Geary et al., J. Pharm. Exp. Therap., 2001, 296, 890-897). Despite the ability of current antisense compounds to be delivered to the kidney, there is a need for development of improved antisense compounds that effect target mRNA reduction in the kidney at lower doses and without toxicity.
The kidney is an important target for antisense therapeutics due to its role in controlling many metabolic processes. A number of genes expressed in the kidney have been associated with the development of metabolic disease. Two such examples are the sodium-dependent glucose cotransporter 2 (SGLT2) and connective tissue growth factor (CTGF), both of which have been linked to the development and/or progression of diabetes.
Sodium-Dependent Glucose Cotransporter 2 (SGLT2)
Diabetes is a disorder characterized by hyperglycemia due to deficient insulin action. Chronic hyperglycemia is a major risk factor for diabetes-associated complications, including heart disease, retinopathy, nephropathy and neuropathy. As the kidneys play a major role in the regulation of plasma glucose levels, renal glucose transporters are becoming attractive drug targets (Wright, Am. J. Physiol. Renal Physiol., 2001, 280, F10-18).
Synthetic agents that are derived from phlorizin, a specific inhibitor of sodium/glucose transporters, have been designed and include T-1095, and its metabolically active form T-1095A (Tsujihara et al., J. Med. Chem., 1999, 42, 5311-5324). Phlorizin, T-1095 and T-1095A all inhibited sodium-dependent glucose uptake in brush border membranes prepared from normal and diabetic rat kidney, rat small intestine, mouse kidney and dog kidney, as well as in Xenopus oocytes injected with human SGLT mRNA (Oku et al., Diabetes, 1999, 48, 1794-1800; Oku et al., Eur. J. Pharmacol., 2000, 391, 183-192). These agents have been tested as antidiabetic compounds in laboratory animals with genetic and streptozotocin-induced diabetes. In these models, administration of these compounds inhibited renal SGLT activity, increased urinary glucose excretion and improved glucose tolerance, hyperglycemia and hypoinsulemia (Arakawa et al., Br. J. Pharmacol., 2001, 132, 578-586; Oku et al., Diabetes, 1999, 48, 1794-1800; Oku et al., Eur. J. Pharmacol., 2000, 391, 183-192). Prolonged treatment of db/db mice with T-1095 yielded similar results and also almost completely suppressed the increase of urinary albumin and improved renal glomeruli pathology, indicating a beneficial influence on renal dysfunction and a protective effect against nephropathy, respectively (Arakawa et al., Br. J. Pharmacol., 2001, 132, 578-586). Diabetic nephropathy is the most common cause of end-stage renal disease that develops in many patients with diabetes. In Zucker diabetic fatty rats, long-term treatment with T-1095 lowered both fed and fasting glucose levels to near normal ranges. Also observed were recovered hepatic glucose production and glucose utilization rates without a significant improvement in skeletal muscle glucose utilization rate, indicating that hyperglycemia contributes to insulin resistance in hepatic and adipose tissue in this rat model of diabetes. These results further suggest that glucotoxicity, which results from long-term hyperglycemia, induces tissue-dependent insulin resistance in diabetic patients (Nawano et al., Am. J. Physiol. Endocrinol. Metab., 2000, 278, E535-543).
Other SGLT2 inhibiting compounds are known in the art, such as the c-aryl glucosides disclosed and claimed in U.S. Pat. No. 6,414,126, which are inhibitors of sodium dependent glucose transporters found in the intestine and kidney and are proposed to treat diabetes, hyperglycemia and related diseases when used alone or in combination with other antidiabetic agents (Ellsworth et al., 2002).
The US pre-grant publication 20030055019 claims and discloses isolated mutant proteins selected from a group which includes SGLT2, the corresponding nucleic acid molecules encoding said mutant proteins, isolated antisense derivatives of the nucleic acid sequences encoding said mutant proteins, as well as methods of delivering said antisense nucleic acid derivatives to treat or prevent hypertension, diabetes, insulin sensitivity, obesity, dyslipidemia and stroke. This application also discloses the antisense molecules may be DNA or RNA or a chimeric mixture, single-stranded or double-stranded or may comprise a ribozyme or catalytic RNA (Shimkets, 2003).
The European Patent Applications EP 1 293 569 and EP 1 308 459 claim and disclose a polynucleotide comprising a protein-coding region of the nucleotide sequence of any one of a group of sequences which includes a nucleic acid sequence encoding human SGLT2, an oligonucleotide comprising at least 15 nucleotides complementary to the nucleotide sequence or to a complementary strand thereof and an antisense polynucleotide against the claimed polynucleotide or a part thereof. These applications disclose the use of said antisense polynucleotides for suppressing the expression of a polypeptide of the invention and for gene therapy (Isogai et al., 2003, Isogai et al., 2003).
Although phlorizin and its derivatives are potent inhibitors of sodium-glucose cotransporters, these agents do not specifically inhibit a single species of SGLT, thus all SGLTs in all tissues are affected. Thus, there remains a need for therapeutic compounds that target specific SGLT species. Antisense technology is an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic and research applications for the modulation of SGLT2 expression. Furthermore, given the role of SGLT2 in the development of diabetes, antisense compounds with the ability to be delivered to the kidney and specifically inhibit SGLT2 are highly desirable.
Connective Tissue Growth Factor (CTGF)
Connective tissue growth factor (CTGF; also known as ctgrofact, fibroblast inducible secreted protein, fisp-12, NOV2, insulin-like growth factor-binding protein-related protein 2, IGFBP-rP2, IGFBP-8, HBGF-0.8, Hcs24, and ecogenin) is a member of the CCN (CTGF/CYR61/NOV) family of modular proteins, named for the first family members identified, connective tissue growth factor, cysteine-rich (CYR61), and nephroblastoma overexpressed (NOV), but the family also includes the proteins ELM-1 (expressed in low-metastatic cells), WISP-3 (Wnt-1-induced secreted protein), and COP-1 (WISP-2). CCN proteins have been found to be secreted, extracellular matrix-associated proteins that regulate cellular processes such as adhesion, migration, mitogenesis, differentiation, survival, angiogenesis, atherosclerosis, chondrogenesis, wound healing, tumorigenesis, and vascular and fibrotic diseases like scleroderma (Laui and Lam, Exp. Cell Res., 1999, 248, 44-57).
Connective tissue growth factor is expressed in fibroblasts during normal differentiation processes that involve extracellular matrix (ECM) production and remodeling, such as embryogenesis and uterine decidualization following implantation. Connective tissue growth factor is also frequently overexpressed in fibrotic skin disorders such as systemic sclerosis, localized skin sclerosis, keloids, scar tissue, eosinophilic fasciitis, nodular fasciitis, and Dupuytren's contracture. Connective tissue growth factor mRNA or protein levels are elevated in fibrotic lesions of major organs and tissues including the liver, kidney, lung, cardiovascular system, pancreas, bowel, eye, and gingiva. In mammary, pancreatic and fibrohistiocytic tumors characterized by significant connective tissue involvement, connective tissue growth factor is overexpressed in the stromal compartment. In many cases, connective tissue growth factor expression is linked spatially and temporally to the profibrogenic cytokine transforming growth factor-beta (TGF-β) (Moussad and Brigstock, Mol. Genet. Metab., 2000, 71, 276-292).
Expansion of ECM with fibrosis occurs in many tissues as part of the end-organ complications of diabetes (i.e. diabetic nephropathy), and advanced glycosylation end products (AGE) are implicated as one causitive factor in diabetic tissue fibrosis. In addition to being a potent inducer of ECM synthesis and angiogenesis, connective tissue growth factor is increased in tissues from rodent models of diabetes. AGE treatment of primary cultures of CRL-2097 and CRL-1474 nonfetal human dermal fibroblasts resulted in an increase in steady state levels of connective tissue growth factor mRNA as well as protein levels in conditioned medium and cell-associated connective tissue growth factor, while other IGFBP-related proteins were not upregulated by AGE. Thus, AGE upregulates the profibrotic and proangiogenic protein connective tissue growth factor, which may play a role in diabetic complications (Twigg et al., Endocrinology, 2001, 142, 1760-1769).
Connective tissue growth factor has been associated with the development of diabetes-related conditions, including diabetic nephropathy. Diabetic nephropathy is a common complication in patients with either type 1 or type 2 diabetes mellitus and is recognized to cause severe morbidity and mortality. Structural hallmarks of advanced diabetic nephropathy are glomerulosclerosis and tubulointerstitial fibrosis leading to kidney failure. Current therapies include ACE inhibitors and angiotensin II receptor blockers, both of which are not justified for blanket use among all newly diagnosed patients since only 30-40% will develop progressive renal disease and the long term side effects of these drugs are unknown.
In addition to the need for safe and effective treatments for diabetes is a need for a reliable method to accurately predict, at early stages of disease, which diabetic patients will develop nephropathy and progress to kidney failure. Persistent microalbuminuria is regarded as a predictor of established vascular damage and an indicator of incipient nephropathy. Studies of renal biopsies from patients with type 1 diabetic nephropathy demonstrate an increase in expression of CTGF in renal tissue exhibiting microalbuminuria and nephropathy, relative to normal control tissues (Adler et al., Kidney Int., 2001, 60, 2330-2336), suggesting CTGF is not only a mediator of diabetic nephropathy, but could be used as a marker for the development of disease (Riser et al., Kidney Int., 2003, 64, 451-458).
Disclosed and claimed in U.S. Pat. No. 5,876,730 is a substantially pure or isolated polypeptide characterized as having an amino acid sequence corresponding to the carboxy terminal amino acids of a connective tissue growth factor (CTGF) protein, wherein the polypeptide has an amino acid sequence beginning at amino acid residue 247 or 248 from the N-terminus of connective tissue growth factor, an isolated polynucleotide sequence encoding the connective tissue growth factor polypeptide, a recombinant expression vector which contains said polynucleotide, a host cell containing said expression vector, and a pharmaceutical composition comprising a therapeutically effective amount of connective tissue growth factor polypeptide in a pharmaceutically acceptable carrier. Antisense oligonucleotides are generally disclosed (Brigstock and Harding, 1999).
Disclosed and claimed in U.S. Pat. Nos. 5,783,187; 5,585,270; 6,232,064; 6,150,101; 6,069,006 and PCT Publication WO 00/35936 are an isolated polynucleotide encoding the connective tissue growth factor polypeptide, expression vectors, host cells stably transformed or transfected with said vectors; an isolated polynucleotide comprising 5′ untranslated regulatory nucleotide sequences isolated from upstream of connective tissue growth factor, wherein said untranslated regulatory nucleotide sequences comprises a transcriptional and translational initiation region and wherein said sequence is a TGF-beta responsive element; an isolated nucleic acid construct comprising a non-coding regulatory sequence isolated upstream from a connective tissue growth factor (CTGF) gene, wherein said non-coding regulatory sequence is operably associated with a nucleic acid sequence which expresses a protein of interest or antisense RNA, wherein said nucleic acid sequence is heterologous to said non-coding sequence; and a fragment of connective tissue growth factor (CTGF) polypeptide having the ability to induce ECM synthesis, collagen synthesis and/or myofibroblast differentiation, comprising an amino acid sequence encoded by at least exon 2 or exon 3 of said polypeptide. Further claimed is a method for identifying a composition which affects TGF-beta-induced connective tissue growth factor expression, and a method of diagnosing a pathological state in a subject suspected of having a pathology selected from the group consisting of fibrotic disease and atherosclerosis, the method comprising obtaining a sample suspected of containing connective tissue growth factor, whereby detecting a difference in the level of connective tissue growth factor in the sample from the subject as compared to the level of connective tissue growth factor in the normal standard sample is diagnostic of a pathology characterized by a cell proliferative disorder associated with connective tissue growth factor in the subject. Further claimed is a method for ameliorating a cell proliferative disorder associated with connective tissue growth factor, comprising administering to a subject having said disorder, at the site of the disorder, a composition comprising a therapeutically effective amount of an antibody or fragment thereof that binds to connective tissue growth factor, wherein said antibody or fragment thereof does not bind to PDGF. Antisense oligonucleotides are generally disclosed (Grotendorst, 2000; Grotendorst and Bradham, 2001; Grotendorst and Bradham, 2000; Grotendorst and Bradham, 1996; Grotendorst and Bradham, 1998; Grotendorst and Bradham, 2000).
Disclosed and claimed in PCT Publication WO 99/66959 is a device for promoting neuronal regeneration, comprising a gene activated matrix comprising a biocompatible matrix and at least one neuronal therapeutic encoding agent having an operably linked promoter device, wherein the neuronal therapeutic encoding agent encodes an inhibitor of neuronal cell growth, and wherein the inhibitor of neuronal cell growth is selected from the group consisting of NFB42, TGF-beta, connective tissue growth factor (CTGF), and macrophage migration inhibitory factor (MIF), and wherein the neuronal therapeutic encoding agent is selected from the group consisting of a nucleic acid molecule, a vector, an antisense nucleic acid molecule and a ribozyme (Baird et al., 1999).
Disclosed and claimed in PCT Publication WO 00/27868 is a substantially pure connective tissue growth factor polypeptide or functional fragments thereof, an isolated polynucleotide sequence encoding said polypeptide, said polynucleotide sequence wherein T can also be U, a nucleic acid sequence complementary to said polynucleotide sequence, and fragments of said sequences that are at least 15 bases in length and that will hybridize to DNA which encodes the amino acid sequence of the connective tissue growth factor protein under moderate to highly stringent conditions. Further claimed is an expression vector including said polynucleotide, a host cell stably transformed with said vector, an antibody that binds to said polypeptide, and a method for producing said polypeptide. Further claimed is a method for inhibiting the expression of connective tissue growth factor in a cell comprising contacting the cell with a polynucleotide which binds to a target nucleic acid in the cell, wherein the polynucleotide inhibits the expression of connective tissue growth factor in the cell, wherein the polynucleotide is an antisense polynucleotide, as well as a kit for the detection of connective tissue growth factor expression comprising a carrier means being compartmentalized to receive one or more containers, comprising at least one container containing at least one antisense oligonucleotide that binds to connective tissue growth factor (Schmidt et al., 2000).
Disclosed and claimed in PCT Publication WO 00/13706 is a method for treating or preventing fibrosis, the method comprising administering to a subject in need an effective amount of an agent that modulates, regulates or inhibits the expression or activity of connective tissue growth factor or fragments thereof, and wherein the agent is an antibody, an antisense oligonucleotide, or a small molecule. The method is directed to treating kidney fibrosis and associated renal disorders, in particular, complications associated with diabetes and hypertension (Riser and Denichili, 2000).
Disclosed and claimed in PCT Publication WO 01/29217 is an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from a group comprising NOV1, NOV2 (connective tissue growth factor), and NOV3, a mature form or variant of an amino acid sequence selected from said group, as well as a nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from said group as well as mature and variant forms or fragments of said polypeptides, and the complement of said nucleic acid molecule. Antisense oligonucleotides are generally disclosed (Prayaga et al., 2001).
A phosphorothioate antisense oligonucleotide, 16 nucleotides in length and targeted to the translation initiation start site, was used to inhibit expression of connective tissue growth factor and suppress proliferation and migration of bovine aorta vascular endothelial cells in culture (Shimo et al., J. Biochem. (Tokyo), 1998, 124, 130-140). This antisense oligonucleotide was also used to show that connective tissue growth factor induces apoptosis in MCF-7 human breast cancer cells and that TGF-beta-induced apoptosis is mediated, in part, by connective tissue growth factor (Hishikawa et al., J. Biol. Chem., 1999, 274, 37461-37466). The same antisense oligonucleotide was also found to inhibit the TGF-beta-mediated activation of caspase 3 and thus to inhibit induction of TGF-beta-mediated apoptosis in human aortic smooth muscle cells (HASC) (Hishikawa et al., Eur. J. Pharmacol., 1999, 385, 287-290). This antisense oligonucleotide was also used to block connective tissue growth factor expression and demonstrate that high blood pressure upregulates expression of connective tissue growth factor in mesangial cells, which in turn enhances ECM protein production and induces apoptosis, contributing to the remodeling of mesangium and ultimately glomerulosclerosis (Hishikawa et al., J. Biol. Chem., 2001, 276, 16797-16803).
Currently, there are no known therapeutic agents that effectively inhibit the synthesis of connective tissue growth factor and thus far. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting connective tissue growth factor function, which for the treatment of diseases like diabetes, requires compounds that can be effectively delivered to the kidney.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of connective tissue growth factor expression.
The present invention provides antisense compounds and methods for optimized kidney targeting as well as methods for preventing diseases and conditions associated with expression of selected target genes in the kidney. Further provided are compounds and methods for modulating blood glucose levels and for inhibiting the development of diabetic nephropathy. Also provided are compounds and methods for modulating SGLT2 and CTGF expression in kidney cells and tissues.